Anticipating Challenges in Optical Nanobiosensors for Global Detection of Respiratory Viruses and Emerging Threats

The unprecedented SARS-CoV-2 pandemic has opened huge opportunities for nanomaterial-based biosensors focused on timely detection of emerging respiratory viruses, where challenges must address actions for fast response and massive application. Accordingly, we provide a comprehensive perspective on critical aspects, including nanomaterials, biofunctionalization strategies, and bioreceptors engineering to increase accuracy, emphasizing optical nanobiosensors. The first biosensing prototype performance reveals the need to consider crucial factors for improvement, such as handling detection in complex matrices, standardization for commercial purposes, portability, integration with artificial intelligence, sustainability, and economic feasibility. By achieving these goals, biosensors would foster a prepared global healthcare landscape.

Biosensors have revolutionized healthcare by enabling the recognition and quantification of biological analytes.These are integrated receptor-transducer devices composed of a biorecognition element that can be attached to functionalized materials adapted for detecting target analytes.The coupled transducer converts this interaction into an output signal, which can be processed to provide a quantifiable result.Fabricating biosensors involves different stages such as material characterization, surface functionalization, immobilization of biorecognition elements, assessment of transduction responses (optical, electrochemical, piezoelectric), and output-signal processing.Moreover, biosensing devices must meet the ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end users) to be efficient tools for diagnostics. 1The progress in modern biosensors (4th generation modules) owes much to nanomaterials due to their unique properties, including high surface area-to-volume ratio, tunable optoelectronic properties, and biocompatibility. 2,3anomaterials have the potential to evolve biosensors in more sensitive, selective, and stable devices, converging in higher applicability in real environments.The last SARS-CoV-2 pandemic has established an inflection point in future developments, with much to explore for highly transmissible viruses.Respiratory viruses such as adenovirus, rhinovirus, coronaviruses (SARS, SARS-CoV-2), human metapneumovirus, and the different types of influenza are causative agents of disease in humans that can evolve in severe infections, with efficient adaptation, high transmission rate, and high mutational frequency. 4Hence, accurate and fast identification is a priority to avoid rapid dissemination, which has been standardly confronted by biomolecular techniques like the quantitative realtime Polymerase Chain Reaction (qPCR).Currently, these strategies offer multiplexed and faster portable versions (<1 h), compatible with the Point-of-Care (POC) approach. 5,6However, these options become unaffordable for massive testing since they require expensive reagents and equipment.Conversely, the feasibility of rapid tests based on optical immunoassays was shown, representing a practical self-testing alternative for final users.Almost four years after the start of the COVID-19 outbreak, multiple approaches for rapid testing biosensors were introduced and commercialized, contributing to medical decisions, but there are still concerns about conceptualization for end-consumer use and how to ensure detection accuracy.The present revision explores the advancements, challenges, needs, and future directions for each stage involved in developing optical nanobiosensors for respiratory viruses to reach accuracy and affordability for widespread use.

Current Status in Advancing Biosensing Devices for Respiratory Virus Detection
Nanomaterials as biosensor platforms.-Theworldwide intervention for managing the SARS-CoV-2 emergency allowed a fast implementation of biosensors within the last three years (2020-2023), compared to influenza and other respiratory viruses (more than two decades without diversified research on biosensing).Figure 1 depicts the main stages that have elapsed from the pandemic outbreak until the implementation of SARS-CoV-2 testing devices.Multidisciplinary research facilitated the identification of virus etiology through medical diagnosis, whereas omic techniques allowed the sequencing of virus genomes and the identification of membrane proteins, receptors, and target sites in their hosts (Fig. 1a).All this research supported processing biorecognition molecules (bioreceptors) to give different biosensor proposals (Fig. 1b).This expansion has been conceivable thanks to integrating nanomaterials, where a proper characterization at the nanoscale has the advantage of improving device sensitivity and limits of detection (LODs).Nanomaterials can acquire different morphologies with a high surface-to-volume ratio for efficient conjugation with biomolecules, improving their interaction at lower concentrations, offering variable responses in transduction, and enhancing output signals. 7][10] For instance, gold, silver, platinum, and graphene are still the most used nanomaterials for electrochemical biosensors. 11Furthermore, trends to improve signal transduction include developing nanocomposites, metallic nanoparticles, screen-printed electrodes, nanoelectromechanical systems (NEMS), incorporating organic conductive polymers and QDs. 12,13Two-dimensional materials (2DMs) have also been immersed in electrochemical sensing for influenza H9N2 using reduced oxide graphene (rGO), GO for H1N1 influenza virus, and graphene for SARS-CoV-2 identification. 14onetheless, achieving stable measurements in complex samples (saliva, blood, serum, food) is challenging for electrochemical devices due to matrix interferences that generate background noise and lack of reproducibility, limiting their accuracy. 12therwise, optical nanobiosensors have opened new opportunities for developing portable and easy-to-use devices to provide faster results without specialized equipment; 15 their advancements z E-mail: adutt@iim.unam.mx;rasd@iim.unam.mxECS Sensors Plus, 2023 2 044601 regarding respiratory viruses date from almost 30 years ago, contributing to influenza detection through Surface Plasmon Resonance (SPR). 16Besides, nanomaterials such as metal and metal oxide nanoparticles (gold, silver, platinum, graphene), polymers, optical fibers, carbon nanotubes, 17 QDs, 18 and ZnO nanomaterials 19 have been used for virus and SARS-CoV-2 rapid biosensing 20 by colorimetric assays, SPR, Surface-Enhanced Raman Scattering (SERS), surface-enhanced fluorescence (SEF), and surface-enhanced infrared absorption spectroscopy (SEIRA). 8,15The ultimate advances involve using QDs and semiconductor nanocrystals that provide brightness and photostability.Their emission can be finely tuned based on size, resulting in high quantum yield and narrow emission spectra suitable for colorimetric and fluorescent biosensing devices, even in microfluidic systems. 21rom now on, our discussion will be centered on the viability of optical nanobiosensors for detecting respiratory viruses, emphasizing current perspectives on affordability for massive testing and self-diagnostics.
Overview of nanomaterial biofunctionalization strategies.-Extensiveresearch to increase nanomaterials' compatibility has been performed employing cross-linkers, labels, site-direction of bioreceptors, covalent-binding, and signal-response enhancers to accomplish the immobilization of diverse biomolecules (antibodies, enzymes, DNA/RNA probes, aptamers, peptides, among others).Optimized strategies rely on parameters such as probe density, orientation, and self-assembly monolayers (SAMS), which can be modified to reach maximum attachment efficiency, depending on the components' nature.Optical biosensor measurements are based on how the incident light interacts between the bioreceptor and the target.Therefore, it is vital to consider the density of immobilized biomolecules, which must be reproducible due to the changes in dispersion, wavelength, refractive index, or absorption that modify the optical signal. 22SAMS is the primary strategy to perform biofunctionalization, allowing the formation of ordered molecular assemblies through the adsorption of active surfactants on the surface.Since the head group reacts chemically with the substrate, 23 using alkyl silanes, alkanoic acids, and thiols is the best option.Nonetheless, conditions such as concentration of reactants to achieve homogeneous monolayers, physical (O 2 plasma) or chemical activation of nanomaterials (wet-chemistry for OH - formation or bimolecular nucleophilic substitution-SN 2 mechanism), and the control of self-assembly (by temperature or non-polar solvents) must be handled without affecting the optical response (fluorescence or photoluminescence), which represent significant challenges at the nanoscale. 24he immobilization step determines the sensitivity and selectivity of biosensors.Biomolecule attachment can be performed by two strategies: the random type, which could require or not linkers after SAMS for the union of biological elements; nevertheless, the lack of orientation in active sites of recognition (antibodies, enzymes, and aptamers) limits their sensitivity.The second strategy focuses on site-direction of bioreceptors to keep the active sites available for recognition.For this, different cross-linkers such as Glutaraldehyde, N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (SNHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 24 could be used to enhance sensitivity.The use of linker molecules should be focused on achieving high homogeneity in surface activation, evaluating critically before choosing one method or another to avoid steric hindrance and interferences in signal transduction.In addition, it must be assessed the cost-benefit of advantages that justify the use of these linkers and the blockage of nonspecific sites with inert proteins such as BSA, which could cause adverse effects of steric hindrance.Moreover, innovations in chemical and/or synthetic ECS Sensors Plus, 2023 2 044601 molecules constitute an emergent field that must be further exploited since they provide advantages such as high stability, mimetic activities to emulate natural biomolecules, and low-cost synthesis.
Current trends on portable and easy-handle devices for optical response.-Influenzaviruses were the former pathogens that sent the basis for the biosensing of respiratory diseases, incorporating the principle of immunoassays on Lateral flow assay (LFA) and Dual Recognition Element LFA (DRELFA).This enabled the fabrication of the first commercial rapid tests as POC devices that showed a fast detection of H1N1 25 and H3N2. 26These strategies pioneered the development of SARS-CoV-2 rapid tests during the recent pandemic.Furthermore, using nanozyme probes as nanoparticles, strips, nanocomposites, bioconjugates, and nano-complexes has contributed significantly to the fabrication of sensitive tests for pathogen diagnosis based on fast visual and colorimetric changes. 27For instance, nanozyme biosensors based on Au nanoparticles were developed for avian influenza virus H5N1, 28 and murine norovirus (MNV) recognition through peroxidase-like Au nanoparticle aptasensors. 29Recently, Meng.et al. reported FeS 2 nanozyme-strips for nucleic detection of SARS-CoV-2, achieving high sensitivity comparable with RT-PCR, demonstrating the suitability of the catalytic activity of nanozymes. 30On the other hand, microfluidic technology has enhanced automation portability at a microvolume scale, allowing low LODs and successful integration of biosensors with computational programming.Microfluidics has contributed to the identification of different types of influenza viruses, 31 human adenovirus (HAdV), 32 and SARS-CoV-2, representing alternatives for fast detection (<40 min), maintaining accuracy even with clinical human samples. 32,33Moreover, multiplex devices are rightly accomplished by integrating microfluidic chips with different optical responses (fluorescence, luminescence, SPR, SERS, and microring resonators). 34In brief, the current proposals should address the costeffectiveness and long-term stability of nanomaterials, bioreceptors, surface functionalization, and transduction response for improved biosensors, ensuring accessibility and self-diagnosis outside lab settings.

Emerging Needs and Future Prospects
As reviewed, research has mainly focused on biosensing of SARS-CoV-2 and influenza.Nevertheless, the acquired experience on nanobiosensors must consider detecting other respiratory viruses like adenoviruses and human metapneumovirus.Figure 2a illustrates a summarized flow chart of the sequential stages with the main perspectives and considerations for developing nanobiosensors with enhanced performance.We show five crucial points for the fabrication of optical biosensing devices: (1) platform-based nanomaterials and biofunctionalization strategies, (2) establishment of LODs and validations, (3) coupling to real matrices and evaluation in clinical samples, (4) versatility and compatibility with intelligent technologies, and (5) economic feasibility.Optical biosensors can find a challenging application for airborne pathogens as they represent high-risk threats for massive infection (Fig. 2b).It is an excellent opportunity for optical approaches because they are suitable for fast analysis of collected particles on filters, ideal for on-site detection and signal transduction based on colorimetric variations, quenching, fluorescence, SERS, SPR, and photonic quantification. 35,36coming directions in platform-based nanomaterials and biofunctionalization strategies.-In the last decade, intense research has been devoted to enhancing LODs and transduction signals, aiming to integrate these data into algorithms and intelligent technologies for smart devices.Even better performance can be attained by enhancing the first stages of prototype fabrication, with tighter control in nanomaterials synthesis, controlling the reproducibility of nanostructured morphologies, surface modification, and immobilization.The first generation of biosensors was focused on planar materials; hence, the established biofunctionalization strategies were SAMS and surface coating. 37Besides, integrating nanotechnology and improving nanomaterials with different morphologies (0D, 1D, 2D, and 3D) reveal the need for updating biofunctionalization strategies.Also, the assessment of nanoscale interactions is critical and necessary to optimize the ongoing process, upgrading as well as the output signals.This could greatly benefit the evaluation of emerging nanomaterials and nanocomposites to be included in biosensor prototypes.In addition, addressing challenges related to stability and reusability is crucial for successfully integrating nanomaterials, as they must retain their properties over time to ensure accuracy and sensitivity.In this context, research groups have turned "Click chemistry" into a versatile alternative to better control nanoparticle bioconjugation due to its high efficiency at room temperature and the achieved stability of conjugated biomolecules due to the small size of ligands. 38As per our knowledge, only three works are related to detecting influenza, 39 syncytial virus, 40 and SARS-CoV-2. 41On the other hand, sustainable materials have the advantage of standing or degrading in the environment without leaving toxic remanents, which in turn reduce the environmental impact on soil and groundwater.Unfortunately, sustainability is not one of the most important aspects considered for biosensor design; however, it should be essential for disposable biosensing prototypes for massive detection.In this context, materials such as graphene, cellulose, and paper-based biosensors are suggested as sustainable approaches with optical and naked-eye response signals (LFA) for SARS-CoV-2 detection. 42Regarding biorecognition molecules, a literature survey (Web of Science, 2023) exhibits that antibody-based devices are the prevalent biosensing strategy to detect respiratory viruses (nearly 68%), followed by DNA probes (12%), enzymes (11%), and aptamers (9%).Devices based on DNA probes offer high stability, affinity, and easy attachment over the surface, improving LODs. 43Likewise, aptamers are less expensive than antibodies, providing stability, reusability, and long-term storage. 22Accordingly, employing these kinds of bioreceptors is a perspective for valuable consideration.Currently, synthetic biology also boosts the employment of molecules of artificial nature (labeled or label-free), facilitating the fabrication of improved biosensing devices that can withstand harsh biological conditions.[46] Establishment of LODs and standardization advances.-Atthis time, the efficient performance of biosensors is evaluated by quantifying LODs, which directly correlates with sensitivity.Although most available research includes this evaluation, no general convention or criteria exist for LODs measurement.This means that standardization of protocols for the preparation of biological receptors (concentration, immersion matrix) is necessary and must be explicitly established for each type of biosensor (optical, electrochemical, piezoelectric) for a particular configuration (nanostructured, 0D, 1D, 2D, 3D, composites).It would give certainty with respect to the variations of signal response considered as significant and determination of signal-to-noise ratio compared to the appropriate controls.Nevertheless, the International Organization for Standardization (ISO) supports certain stages involving nanotechnology use, nano-object assembly, surface modification, characterization, and measurement standards (https://www.iso.org/standards.html).The following ISO laws correlate with the contents described here for nanomaterial biosensing applications: 1. ISO/TR 14187:2020(en) Characterization of nanostructured materials, 2. ISO/TR 19693:2018(en) Characterization of functional glass substrates for biosensing applications, 3. ISO/TS 21412:2020 (en) Nano-object-assembled layers for electrochemical bio-sensing applications.Specification of characteristics and measurement methods, 4. ISO 22870:2016 Point-of-care testing (POCT): Requirements for quality and competence.Mainly, ISO/TS 21412:2020 for electrochemical devices comprises the specifications ECS Sensors Plus, 2023 2 044601 for measurement of nano-objects assembled layer on electrode surfaces for sensing applications, allowing a homologation in performance and qualifications for nano-object-modified electrodes.However, for optical biosensors, there is still a lack of a focused methodology that includes all the specifications required for evaluation, analysis, and standardization for commercial purposes.Instead, there are recent ISO laws that could serve as a reference for quantitative analysis: 1. ISO 24421:2023(en) Minimum requirements for optical signal measurements in photometric methods for biological samples, 2. ISO 24465:2023(en) Determination of the minimum detectability of surface plasmon resonance device.
Since different conditions are intensively tested by research teams worldwide, universal standards are necessary to consider an expedited application of optical biosensing prototypes in diverse countries.
Enhanced detection of nanobiosensors, tackling technical problems.-Theperformance of nanobiosensors is commonly evaluated under laboratory conditions with minimal interferences, obtaining acceptable LODs.Nevertheless, the challenge is reaching optimal signal response in complex matrices, overcoming interferences, and biofouling effects to ensure efficient behavior.To attain this goal, bioassays must be established to assess detection in clinical samples.Recent reports denote an increasing advance in this respect, where electrochemical biosensors have been proved for influenza H1N1 in saliva, 47 influenza H5N1 in chicken serum, 48 norovirus in oyster samples, 49 and SARS-CoV-2 in human serum. 50These responses have been potentiated by using Au nanoparticles as functional transducers, peptides, and DNA probes as biorecognition molecules.On the other hand, optical biosensors have demonstrated suitable responses for sensitive performance in complex samples.For instance, respiratory viruses such as influenza subtypes H3N2, H1N1, H5N1, and norovirus have been detected in human serum, [51][52][53][54][55] influenza H7N9 in human and chicken serum, 56,57 avian influenza H9N2 in chicken lung and liver, 58,59 and SARS-CoV-2 detection in saliva. 60Yet, using antifouling coatings on biosensor surfaces is a novel approach to overcome interferences in detection, low sensitivity, and unspecific adsorption of biological materials (biofouling) found in complex matrices.Antifouling molecules such as electropolymerized polyaniline (PANI) nanowires, 61 terpolymer-brush biointerface, 62 and assembly of polystyrene (PS) beads on gold electrodes 63 have been currently used to increase sensitivity in crude clinical samples, achieving ultra-low LODs.These molecules deserve further analysis for broadening their incorporation in biosensing devices.Finally, applying nanobiosensors for daily use under real-environment conditions must cross the barrier and leap clinical trials to guarantee their safe implementation.Ensure versatility and compatibility with intelligent technologies.-Finally,integrating biosensors with artificial intelligence (AI) and IoT (Internet of Things) holds great promise for widespread use.AI algorithms can analyze and interpret the biosensor data, providing real-time monitoring and feedback.Output signals can be optimized with programming adaptation, microelectronics, machine learning algorithms, 64,65 miniaturization, smartphone operation, and POC devices 66 to create logical and more accurate responses compatible with portable intelligent technology. 60An application is the development of a Graphene-Based Multiplexed immunosensor platform RapidPlex for SARS-CoV-2 diagnosis, presented as a portable and wireless electrochemical platform for ultra-rapid detection in patient blood and saliva samples. 67Since shared information over the internet could be at risk of mishandling and leakage, data privacy must be legally protected and guaranteed for the users of these smart devices.Most of the countries (71%) have legislation for data protection, according to the statistics collected by the United Nations Conference on Trade and Development (2021); however, specific data security regulations must be rigorously established for these forthcoming technologies.Economic feasibility.-One of the most essential aspects of widespread use is the affordability of biosensor proposals; however, there are limitations in this respect.For instance, low concentrations of target analytes are difficult to identify and quantify unless the best materials (in most cases expensive) are used for fabrication.The use of highly pure bioreceptor molecules and the best strategies for biofunctionalization, transduction, and signal amplification are also required.This is the enormous challenge of achieving high efficiencies that can be accomplished by improving available or emergent materials (lowcost composites or unexplored nanoplatforms) and examining their nanoscale interaction with biomolecules.For example, using versatile, practical, and low-cost semiconductor materials such as ZnO 68 offers diverse exploitable fabrication properties for electrochemical 69 or optical biosensors, 70 as reviewed in our previous works. 71In addition, the reversible tuning of ZnO nanowires' photoluminescence from green to blue offers properties like high surface-to-volume ratio and sensitivity to environmental changes for the improvement of optical biosensing platforms, as demonstrated by Galdamez et al., which could be aimed at detecting airborne diseases. 72Interaction with pathogens induces shifts in photoluminescent emission, enabling real-time, labelfree detection. 70This reversibility ensures platform sustainability and the green-to-blue transition adds versatility for further multiplexed configurations.

Conclusions
The SARS-CoV-2 pandemic underscored the vital role of biosensors in global pathogen identification.Even though optical nanobiosensors have offered suitable alternatives for fast and massive detection, challenges still persist, requiring paradigm shifts to reach high specificity and accuracy.Foremost, selecting nanomaterials is one of the fundamental aspects determining optimal biosensing, which must be cost-effective, adaptable, and compatible with bioreceptors.Also, surface activation and bioreceptors' engineering must be directed to output signal enhancement.These former stages can represent building-block approaches for optimization, even before selecting sophisticated signal-amplification systems, as they could greatly influence biosensors' performance in complex matrices, promoting their efficient integration with intelligent technologies.Regulatory issues and homologated criteria for determining LODs must be promptly attended to for the widespread adoption of optical nanobiosensors.

Figure 1 .
Figure 1.Multidisciplinary intervention for the development of SARS-CoV-2 biosensing devices.(a) Overview of the stages arising from the outbreak up to the implementation of biosensors.(b) Trends on SARS-CoV-2 biosensing and directions toward POC devices and portable technologies (Created with Biorender).

Figure 2 .
Figure 2. (a) Schematic representation of the main steps for consideration and optimization of affordable biosensors for widespread use.(b) Perspectives on implementing optical biosensors for airborne virus detection (Created with Biorender).
Few works have undertaken this task like the rapid tests for COVID-19 diagnosis: COR-DIAL-1 Portable and Connected Biosensor (France), The Abbott Panbio™ COVID-19 Ag Rapid Test (Switzerland) and TestNPass (IVDMD) for CoViD-19 Diagnosis on Saliva Sample (GraphealNpas) (France), which can be found at the web page of ClinicalTrials.gov(U.S. National Library of Medicine).